The long-range goals of this proposal can best be placed into two major categories: (1) the acquisition of isotope labelled, UV-photo-affinity analogs of regulatory nucleotides (cAMP and cGMP) and (2) the utilization of these highly specific probes to analyze the nucleotide-protein receptor interaction in various stages of the spermatozoan's life history. The isotope labelled, UV-photo-affinity analogs (8-azido) of adenosine and guanosine have been synthesized by Dr. Boyd Haley (8N3-cAM32P and 8N3-cAM32P). They are known to physiologically mimic regular cAMP and cGMP and to bind to their respective receptors. The analogs exhibit saturation and competition kinetics. By various standard biochemical techniques (slab gel electrophoresis, and radiographic analysis, and scintillation counting) the identity of the receptors is being resolved in a number of different systems. Our preliminary data support the contention that we have labelled and begun the identification and characterization of the mammalian spermatozoan cAMP receptor. Thus the first goal of this research is to increase our synthesis activity of these analogs so that we can expand our utilization of these analogs in the overall characterization of cAMP and cGMP spermatozoa receptors and analysis of related nucleotide biochemistry. To pursue the second goal of this proposal we intend to study both surface and lysed speramtozoa membrane preparations for their cAMP and cGMP receptors by use of these analogs. Ejaculated spermatozoa of mammalian (dog, rabbit, and human) and invertebrate (sea urchin) spermatozoa will be analyzed for their receptors. Where appropriate comparisons between various epididymal fractions will be made to the ejaculated fractions. Furthermore, spermatozoa will be fractionated into head, mid-piece, and tail for analysis of cAMP and cGMP receptors on these respective cellular fractions. Comparisions between receptors on the head, mid-piece and tail will be made with further subcellular spermatozoa fractions. Analysis of the identity of cAMP and cGMP receptors will be pursued in relation to the development of spermatozoa motility and capacitation. The emphasis of this proposal is to blend basic biochemical and physiological analysis of spermatozoa nucleotide receptors to fundamental problems of fertility.